Coxsackievirus and adenovirus receptor (CAR) is a member of the Ig-type superfamily of cell adhesion molecules. In polarized epithelial cells CAR is expressed at the tight junction. The mouse CAR gene is composed of at least eight exons, and CAR splice variants that differ at the end of the cytoplasmic tail have been identified in a number of tissues. The present study aimed to examine the expression of (CAR), a TJ protein sealing the muticellular contact point during preimplantation embryos and role of CAR in the formation and integrity of the blastocoel. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG in oviducts and uterus, and we also obtained gestation day 5 and 6.5 embryos in uterus. All samples were subjected to RT-PCR, immunofluorescence and immunohistochemistry analysis. To analyze epithelial permeability of CAR, we examined permeability of FITC-labeled dextran (MW 40 kDa) following functional blocking of CAR in blastocysts. Long isoform of CAR mRNA was expressed from throughout the preimplantation development and markedly increased at morulae stage onward. Small amount of short isoform CAR mRNA was expressed at blastocyst stage. On Western blot, 64 kDa protein was detected together with 43 kDa protein corresponding to short and long forms CAR, respectively in blastocysts. CAR immunoreactivity was found in cell contacts between blastomeres from 4-cell stage onward. Under Ca2+ switching condition blocking antibodies for CAR increased the permeability of blastocysts to FITC-dextran, a permeability tracer. At 5 dpc, trophoblasts of the implanting embryos were immunoreactive with anti-CAR IgG. At 6.5 dpc, the egg cylinder stage in mouse, the visceral and parietal endoderm were immunoreactive with anti-CAR IgG. Our results suggest that alternative splicing of CAR transcript is highly dependent on the development of expanding blastocyst. CAR may play a crucial role as a barrier to adenovirus infection and adhesion molecule for epithelial permeability during peri-implantation period.